Pulsed acoustofluidic pipetting: An automated platform mimicking manual pipetting for accelerated cell staining

Accurate and efficient cell staining is a fundamental prerequisite for reliable cell analyses. Conventional antibody labeling methods primarily rely on passive diffusion or manual pipetting—a widely used yet labor-intensive and inconsistent mixing technique. Manual pipetting, while effective in refreshing the local concentration gradient, suffers from operator dependency, poor reproducibility, and potential cell damage due to uncontrolled shear forces. To overcome these limitations, we developed a Pulsed Acoustofluidic Pipetting (PAP) method that mimics and automates the manual pipetting process using localized acoustofluidic actuation within standard microcentrifuge tubes. The system employs programmable acoustic pulses to generate periodic micro-vortices that actively refresh the concentration boundary layer, thereby accelerating antibody–antigen binding while maintaining a gentle microenvironment. A compact acoustofluidic module was built to integrate automated staining and washing in a single step. Compared with static incubation and manual pipetting, PAP achieved over a twofold increase in staining efficiency within one quarter of the time, with superior uniformity and reproducibility. The method demonstrated consistent enhancement across cell models with high, medium, and low antigen expression, while fully preserving cell viability and fluorescence integrity.